We demonstrated that in the Apc1638N mouse genetic model of intestinal tumorigenesis, introduction of a targeted inactivation of a single p2iWAF1/clp1 allele was sufficient to both enhance tumor formation and eliminate the ability of sulindac to inhibit tumor formation (Yang et al, Cancer Res, 61, 565, 2001 a; Yang et al, Cancer Res. 61, 6297, 2001 b). We have newfound that the reason for the apparent haplo-insufficiency of p21 in both tumor suppression, and in the chemopreventive response to sulindac, is because the remaining wild-type p21 allele is functionally inactivated by methylation of CpG island in its promoter. It is therefore not expressed, and cannot be induced by sulindac.
In aim 1, we will determine the mechanism of this inactivation of p21 by promoter methylation by: determining whether the methylation of the wild-type allele in p21 mice is dependent on heterozygosity of the Ape gene; dissecting how the sites of altered methylation in the promoter correspond to cis-acting elements necessary for the response of p21 to sulindac; and assaying the role of altered expression and levels of DNA methyltransferases in the modulation of p21 methylation.
Aim 2 will investigate how selenium, a nutritional preventive agent, reported to cause DNA hypomethylation, and a pharmacological demethylating agent, Decitabine (2'-deoxy-5'-azacytidine), lter methylation of the wild-type allele in the Apc, p21 mice, and restore tumor suppression activity and response to sulindac. Further, we will use gene expression profiling, quantitative real time RT-PCR, and assays of methylation of the Ape, Mlh1 and Muc2 genes, to understand the similarities and differences in the affects of the nutritional and pharmacological agents, and the extent to which altered CpG island methylation at specific loci is translated into altered gene expression.
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